Inhibition of Transcription Initiation buIacRepressor

Initiation of transcription of thelacoperon by RNA polymerase (R) is inhibited by binding oflacrepressor (L) to an operator site which overlaps thelacpromoter (P). We have investigated repression of thelacUV5 promoterin vitrofor a choice of the repressor|mdash|operator binding constant and ranges of thermodynamic activities of L and R which appear to be relevantin vivo. Effects of [L] on the extent of formation and the kinetics of association and dissociation of abortively-initiating open complexes (RPinit) were examined using fluorescence detected abortive initiation and KMnO4chemical probing. The nitrocellulose filter assay was used to measure the dissociation rate constant and the equilibrium constant for binding for L to its operator site in the absence of R. For the chosen solution conditions, we find that both the observed velocity of abortive RNA oligomer synthesis and the KMnO4reactivities of bases in the open region are functions of [L] and [R], demonstrating that formation of both RPinitand the repressor–operator complex (PL) are reversible processes under these conditions, and requiring the use of a relaxation-to-equilibrium analysis to interpret the kinetics. The agreement between dissociation rate constants of RPinitwhen challenged with eitherlacrepressor or heparin, and the dependences on [L] and [R] of abortive synthesis velocities at binding equilibrium and of relaxation rate constants for reversible formation of RPinitfrom PL, all provide evidence for a simple competition mechanism. In this mechanism, and in contrast to recent proposals from other laboratories,lacrepressor inhibits formation of RPinitand hence the observed rate of abortive product synthesis by reducing the equilibrium extent of formation of the first closed complex (RPcl), without affecting either the nature of RPinitor steps in formation of RPinitfrom RPcl.